The longterm objective of this project is to gain an insight into the role of IL-2 in neutrophil activation and to analyze the mechanisms by which such activation occurs. Emerging data from us and others indicate that human neutrophils have the capacity to release a number of cytokines that can influence the immune system. Neutrophils are also present in tumors from patients during both recombinant interleukin-2 (IL-2) cytokine administration and IL-2 gene therapy. We have made the novel observation that human neutrophils constitutively express IL-2R/beta and can be functionally activated via this receptor for antimicrobial activity, tumor necrosis factor (TNF)alpha and IL-8 release. PMN survival is also enhanced by IL-2. The direct activation of neutrophils may thus contribute to tumor rejection during IL-2 cytokine or gene therapy. This proposal will investigate what precisely are the mechanisms which lead to PMN activation by IL-2. First, experiments will be designed to define if all chains of the IL-2R, i.e., alpha, beta and gamma, are present contstitutively or inducible by IL-2. FACS analysis of surface receptors, Western blotting and immunoprecipitation with specific antibodies to each of the 3 IL-2R chains, and Northern blot/RT- PCR analysis of gene expression will be conducted. Co-association of the IL-2R/gamma chain with other cytokine receptors, IL-4, IL-8, G-CSF, will also be investigated. Next, the signalling pathways utilized by PMN in response to IL-2 will be analysed. Western blotting and immunoprecipitation studies using specific antibodies against various phosphotyrosine kinases (PTK), i.e. lck, fyn, lyn, hck, fgr, will lead us to understand which of the src family kinases may be responsible for signal transduction. The mechanism of activation of TNF/alpha and IL-8 gene transcription will also be investigated at the nuclear protein level. The biological relevance of the identified PTK, transcription factors and the various IL-2R will be evaluated. This will be approached by antisense oligonucleotides or by PTK inhibitors for blocking of IL-2 activation of PMN function. Lastly, PMN from patients with X-linked severe combined immunodeficiency will provide the ultimate in vivo confirmation of the role of IL-2R/gamma in PMN function. Such work will bring important insight into the unique interaction of IL-2 with PMN.